ELISA Assay

Written by Administrator

Monday, 23 June 2008

Learn about the ELISA technique. Includes the general steps of ELISA.

ELISA Enzyme Linked ImmunoSorbent Assay

ELISA (Enzyme-Linked ImmunoSorbent Assay) a.k.a: Enzyme ImmunoAssay or EIA, is a common biochemical assay technique used mainly in immunology. This technique is used to detect the presence of an antibody or an antigen in a sample. ELISA is used in medicine and plant pathology as a diagnostic tool as well as a quality control check in various industries. The ELISA technique can be summarised as follows: An unknown amount of an antigen is affixed to a surface, then a specific antibody, which is also linked to an enzyme, is washed over the surface so that it can bind to the antigen. In the final step of ELISA a substance is added which reacts with the enzyme that is linked to the specific antibody which produces a detectable signal. In the case of fluorescence ELISA when light is shone upon the sample any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be measured.

General ELISA technique:

The ELISA technique is dependent on at least one antibody with a specificity for a particular antigen.

1) The sample which contains an unknown amount of antigen is immobilized on a solid support (polystyrene microtiter plate) either via absorption to the surface (non-specifically) or via capture by another antibody specific to the same antigen, i.e. "sandwich" ELISA (specifically).

2) After when the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be either covalently linked to an enzyme or it can itself be detected by the introduction of a secondary antibody which is linked to an enzyme through bioconjugation.

3) Between each step the plate is washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound.

4) After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal which quantifies the amount of antigen in the sample. ELISA's that are older utilize chromogenic substrates, while newer assays employ flurogenic substrates which also have more sensitivity.